+

WO2005010532A1 - Dosages de liaison multiplexes destines a des reseaux de recepteurs - Google Patents

Dosages de liaison multiplexes destines a des reseaux de recepteurs Download PDF

Info

Publication number
WO2005010532A1
WO2005010532A1 PCT/US2004/019750 US2004019750W WO2005010532A1 WO 2005010532 A1 WO2005010532 A1 WO 2005010532A1 US 2004019750 W US2004019750 W US 2004019750W WO 2005010532 A1 WO2005010532 A1 WO 2005010532A1
Authority
WO
WIPO (PCT)
Prior art keywords
receptor
labeled
compound
array
binding
Prior art date
Application number
PCT/US2004/019750
Other languages
English (en)
Inventor
Ye Fang
Ann M Ferrie
Yulong Hong
Joydeep Lahiri
Fang Lai
Jinlin Peng
Brian L Webb
Original Assignee
Corning Incorporated
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Corning Incorporated filed Critical Corning Incorporated
Priority to JP2006520183A priority Critical patent/JP2007530919A/ja
Priority to EP04776836A priority patent/EP1646871A1/fr
Publication of WO2005010532A1 publication Critical patent/WO2005010532A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the present invention relates to biological, biochemical or chemical binding assays performed on a solid surface.
  • the invention pertains to the use of
  • GPCR G-protein-coupled receptor
  • GPCRs G-protein coupled receptors
  • GPCRs represent the single most important class of drug targets — approximately 50% of current drags target GPCRs; about 20% of the top 50 best selling drugs target GPCRs; more than $23.5 billion in pharmaceutical sales annually are ascribed to medications that address this target class.
  • GPCRs are associated with almost every major therapeutic category or disease class, including pain, asthma, inflammation, obesity, cancer, as well as cardiovascular, metabolic, gastrointestinal and central nervous system diseases.
  • DNA microarrays e.g., Schena, M., et al. "Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray," Science 1995, v. 270, 467-470
  • protein arrays e.g., Mitchell, P., "A Perspective on Protein Microarrays," Nat.
  • protein microarrays are naturally suited for testing compounds against multiple proteins simultaneously, some of the fundamental aspects of multiplexed bioassays using protein chips are yet to be demonstrated.
  • One such fundamental aspect is the need for cocktails of labeled ligands for multiplexed competitive binding assays. Problems due to non-specific binding, cross reactivity, and the lack of general guidelines for choosing labeled ligands have deterred scientists from testing the feasibility of using mixtures of labeled ligands.
  • protein arrays may contain redundant elements — those against which a labeled ligand is not present in the cocktail.
  • the present invention describes the use of receptor microarrays for multiplexed compound profiling or screening involving the use of cocktails of self-assembled labeled ligands.
  • Multi-GPCR target screening based on binding assays requires the use of self-assembled ligand cocktails.
  • the invention also discloses several multiplexed assay formats, including: saturation assays for parallel K d determination, competitive binding assays for parallel IC 50 determination (e.g., selective potency) of a single compound against multiple GPCR targets, or parallel IC 50 determination (e.g., relative potency) of multiple compounds against their corresponding GPCR targets, and competitive binding assays and displacement assays for compound screening.
  • a thematic microarray of GPCRs is made for multiplexed compound screening and profiling.
  • the microarray comprises: a plurality of GPCRs arranged on a substrate at positionally defined locations.
  • all known and orphan GPCRs in human genome
  • a representative index array may include a GPCR member selected from each subfamily of GPCRs.
  • a selectivity panel array may include at least a single GPCR member selected from several related subfamilies of GPCRs.
  • a muta-genesis array may be composed of a GPCR and its variants or mutants, or its corresponding GPCRs originated from different species.
  • the present method and assay format utilizes saturation capabilities of the labeled ligands to bind with their paired receptors for profiling and screening in indirect binding assays which involve competitive binding of a test compound against labeled- ligands.
  • Multi-target binding assays according to the present invention employ cocktails containing labeled ligands capable of self-assembly with their respective receptors.
  • the invention includes a cocktail solution of at least one labeled-ligand.
  • the labeled- ligands in the cocktail solution are selected based on their individual binding ability to each GPCR microspot element in the microarray.
  • Each labeled-ligand should bind to at least one GPCR element with a desired binding affinity (e.g., - 0.1 nM to ⁇ 20 nM) and specificity of at least 50% to 60%, and minimal cross-activity (e.g., ⁇ 10%) with other GPCR elements in the array. Since, multiplexed binding assays are technically challenging for several reasons, this invention addresses potential solutions to problems associated with labeled ligand binding specificity and affinity, assay buffer compatibility, content suitability, detection and data analysis.
  • each microspot in a microarray contains only one kind of probe-receptor.
  • each microspot contains a predominant kind of probe-receptor within a biological membrane, (e.g., a GPCR-membrane preparation obtained from a cell line over expressing the receptor).
  • at least one microspot can contain at least two kinds of detectable probe-receptors.
  • a method to detect the different kinds of receptors involves using simultaneously different labeled ligands with different tags; the tag can be physically or chemically distinguishable from each other.
  • Each labeled ligand binds specifically with one of the detectable receptors.
  • a GPCR array device having a variety of GPCR species associated with the support substrate; such receptor species may include, for instance, neurotensin receptor subtype 1, motilin receptor, delta2 opioid receptor, opioid-like receptor subtype 1, acetylcholine receptor subtype-1 (Ml), and control cell membranes of CHO or HEK cell lines. Additional features and advantageous of the present invention will be revealed in the following detailed description. Both the foregoing summary and the following detailed description and examples are merely representative of the invention, and are intended to provide an overview for understanding the invention as claimed.
  • FIGURE 1 is a schematic representation of an embodiment of the present invention, in which a multiplexed competitive binding assay uses a cocktail solution of labeled ligands with a GPCR microarray.
  • FIGURES 2A-C are schematic representations of a GPCR microarray in a 96-well microplate.
  • FIGURES 3 A-C are concentration dependent total and nonspecific binding graphs showing a multiplexed saturation assay and K d determination of fluorescent ligands using GPCR microarrays.
  • the GPCR microrrays have three receptors: human neurotensin receptor subtype 1 (NTRl), 52 opioid receptor (OP1) and motilin receptor
  • a cocktail solution of three fluorescently-labeled ligands includes: Cy5- neurotensin 2-13 (Cy5-NT) to NTRl, Cy5-naltrexone to OP1, and Bodipy-TMR- motilin 1-16 (BT-MOT) to MOTR.
  • the K values for these three ligands binding with their corresponding receptors in the microarrays were found to be about 2.5 nM for Cy5-NT binding to NTRl, about 1.9 nM for Cy5-naltrexone binding to OP1, and about
  • FIGURES 4A-C are concentration dependent inhibition graphs showing a selective potency determination of naltrexone to delta2 (OP1) (A), MOTR (B) and
  • NTRl in the microarrays against a cocktail labeled ligands (Cy5-NT, BT-MOT and Cy5 -naltrexone).
  • the GPCR microrrays have three receptors, human neurotensin receptor subtype 1 (NTRl), delta2 opioid receptor (OPl) and motilin receptor (MOTR).
  • a cocktail solution of ligands includes three fluorescently-labeled ligands: Cy5-NT to NTRl, Cy5-naltrexone to OPl, and BT-MOT to MOTR. The selective potency of naltrexone to these three receptors is consistent with what reported in literature. Naltrexone is known antagonist to delta2 only.
  • FIGURES 5A-C are concentration dependent inhibition graphs showing a multiplexed potency determination of cocktail compounds to GPCR microarrays using competitive binding assays and a cocktail labeled ligand, according to the present invention.
  • the GPCR microrrays have three receptors, human neurotensin receptor subtype 1 (NTRl), delta2 opioid receptor (OPl) and motilm receptor (MOTR).
  • a cocktail solution of ligands includes three fluorescently-labeled ligands: Cy5-NT to NTRl, Cy5-naltrexone to OPl, and BT-MOT to MOTR.
  • FIGURES 6A and 6B are graphs presenting data for a multiplexed compound screening using multiplexed competitive binding assays, according to the present invention.
  • Figure 6 A shows histograms for selective inhibition of binding of Bodipy- TMR-motilin 1-16 (BT-MOT) and Cy 5 -neurotensin 2-13 (Cy5-NT) in a cocktail solution to a microarray having NTRl and MOT receptors.
  • BT-MOT and Cy5-NT are ligands known to bind to the MOTR and NTRl receptors, respectively.
  • FIG. 6B shows the selective inhibition of the binding of BT-CGP 12177 and Cy5- naltrexone to a microarray of ⁇ l and 52 receptors.
  • BT-CGP can only specifically bind to ⁇ l in the arrays;
  • Cy 5 -naltrexone can only specifically bind to 62 receptor.
  • Naltrexone is an antagonist to 52, while CGP 12177 is a partial agonist to betal receptor.
  • Signals of two channels (Cy3 and Cy5) are examined as a function of different compounds (CGP 12177, naltrexone, and both) at 1 ⁇ M.
  • FIGURES 7A and 7B present data for multiplexed compound screening using single labeled-ligand competitive binding assays according to the present invention.
  • Figure 5A depicts two false-color fluorescence images for selective binding and inhibition to arrays of an adrenergic receptor family.
  • Figure 5 A(i) is a fluorescence image of a microarray having three columns consisting of ⁇ 1, ⁇ 2 and ⁇ 2A adrenergic receptors treated with a solution containing BT-CGP (5 nM).
  • Figure 5 A(ii) is an image of the array after treatment with a solution containing BT-CGP (5 nM) and ICI118551 (10 nM).
  • FIG. 5B is a graph showing histogram analysis of the binding (BT-CGP) and inhibition (in the presence of ICI118551).
  • FIGURES 8A-D show a series of false-color images according to a method by which a displacement assay for compound screening is performed. Multiple arrays of ⁇ l adrenergic receptor, NTRl, and dopamine Dl receptors are used.
  • Figure 8 A and 8B respectively are fluorescence images of a microarray before and after incubation with BT-NT (2 nM).
  • Figure 8C and 8D are fluorescence images showing the displacement of pre-bound BT-NT by unlabeled compounds.
  • FIGURE 9 shows a series of false-color fluorescence images of three columns of microspots in a microarray after the binding of Cy5 -neurotensin 2-13 and BT-motilin 1-
  • FIGURES 10A-D show saturation binding curves of a Cy5-neurotensin 2-13 and
  • FIG. 10A shows the relative binding affinity of BT-Motilin 1-16 to microspots containing MOTR in either the absence (total signal) or presence (non-specific) of a mixture of neurotensin and motilin.
  • Figure 10B is a Scatchard plot of the data shown in Figure 10A.
  • Figure 10C shows the relative binding affinity of BT-
  • Motilin 1-16 to microspots containing the mixture of MOTR and NTRl in either the absence (total signal) or presence (non-specific) of a mixture of neurotensin and motilin.
  • Figure 10D is a Scatchard plot of the data shown in Figure IOC. The Kd value obtained for BT-Motilin 1-16 with MOTR and the mixture of MOTR and NTRl are similar; however, the total binding sites are almost two-fold higher for MOTR alone than those for the MOTR/NTR1 mixture.
  • ligand refers to a chemical molecule or biological molecule that can bind readily to a receptor with a specific binding affinity constant.
  • labeled-ligand refers to either a fluorescently labeled or radioactive isotope-labeled or hapten-labeled (e.g.-biotin) or gold-nano-particle labeled ligand.
  • cocktail solution refers to a medium (e.g., buffered or aqueous solution) having a mixture either of different labeled ligands or of different compounds. Alternatively, in some embodiments, a mixture of both ligands and compounds may be present together in solution.
  • compound refers to a biological molecule, biochemical or chemical entity, molecule, or pharmaceutical drag candidate to be detected.
  • biological molecule or “biomolecule” refers to any kind of biological entity, including, such as, modified and unmodified nucleotides, nucleosides, peptides, polypeptides, proteins, lipids, or saccharides.
  • cognate “corresponding,” or “paired” refers to the reciprocal moiety of a molecule to another; in particular, a ligand that can bind specifically to a given receptor is called a ligand-receptor pair.
  • biospot or “microspot” refers to a discrete or defined area, locus, or spot on the surface of a substrate, containing a biological or chemical probe.
  • receptor microspot refers to a microspot containing a deposit of membrane-bound proteins.
  • the receptor may include a GPCR, a ligand-gated ion channel receptor, a tyrosine kinase receptor, serine/threonine kinase receptor, or guanylate cyclase receptor.
  • GPCR refers to a guanine nucleotide-binding protein-coupled receptor. The GPCR can have either a natural or modified sequence.
  • GPCR membrane or "GPCR membrane fragment” refers to a biological membrane or cell membrane fragment having a GPCR embedded within a membrane layer, or a micelle having a GPCR reconstituted within the micelle.
  • GPCR microspot refers to a microspot containing a deposit of G- protein coupled receptors (GPCRs). The corresponding microspots are referred to as “probe microspots,” and these microspots are arranged in a spatially addressable manner to form a microarray.
  • probe or "receptor probe” refers to a receptor molecule (e.g., GPCR), which according to the nomenclature recommended by B. Phimister (Nature
  • probes are immobilized to a substrate surface.
  • probes are arranged in a spatially addressable manner to form an array of microspots.
  • molecules in the sample selectively and specifically binds to their binding partners (i.e., probes).
  • the binding of a "target" to the probes occurs to an extent determined by the concentration of that
  • substrate or “substrate surface” as used herein refers to a solid or semi-solid, or porous material (e.g., micro- or nano-scale pores), which can form a stable support.
  • the substrate surface can be selected from a variety of materials, including for instance, glass, ceramic, metals, polymers, plastics, or combinations of these.
  • the terai “functionalization” as used herein relates to modification of a solid substrate to provide a plurality of functional groups on the substrate surface.
  • the phrase “functionalized surface” as used herein refers to a substrate surface that has been modified to have a plurality of functional groups present thereon.
  • the surface may have an amine-presenting functionality (e.g., ⁇ -amino-propylsilane (GAPS) coating), or may be coated with amine presenting polymers such as chitosan and poly(ethyleneimine).
  • GAPS ⁇ -amino-propylsilane
  • Ligands for GPCRs are very diverse, including biogenic amines, peptides and proteins, lipids, nucleotides, excitatory amino acids and ions, small chemical compounds, etc. (Morris, A.J., and Malbon, C.C., "Physiological Regulation of G-Protein-linked Signaling," Physiol. Rev. 1999, 79, 1373-1430.)
  • a particular GPCR could couple to one or more trimeric G proteins in a cell line. The binding affinities of agonists to a
  • GPCR depend on the coupling state of the receptor with its G proteins. Compounds that bind with a receptor might have different functionalities, such as agonism, antagonism, super-agonism or inverse agonism. The binding sites involved might be different for different compounds binding to the same receptor. Second, for assay development, buffer compatibility and optimization should be also considered. For example, some GPCR-ligand interactions depend strongly on the presence of particular divalent cations such as Mg 2+ or Mn 2+ . The buffer composition can not only affect the functionality of the membrane proteins, but also can affect the binding affinity of ligands or compounds to the receptors in arrays.
  • the buffer composition also may have a negative impact on the stability and packing of receptor-containing lipid membranes immobilized on the surface, therefore decreasing array performance and assay robustness.
  • the choice of labeled ligands is equally, if not more critical.
  • the ideal labeled-ligand should bind only to its corresponding receptor with high affinity, and with minimal cross reactivity with other receptors in the same microarray, so as to enable the use of the labeled-ligand at low concentrations and to permit stringent washing processes. High affinity, however, is not a requirement for real-time fluorescence measurements.
  • specific properties such as being relatively hydrophilic, having low net charge(s) (preferably, they are positively charged or neutral), good photostability, high binding affinity (low K d , preferably within a nanomolar range of ⁇ 0.5 nM to ⁇ 10 nM), high specificity ( ⁇ 50% or 60%) for a given receptor in the array, and minimal cross-talk to other receptors in the same microarray.
  • nucleotide microarrays e.g.
  • Figure 1 presents a schematic representation of a multiplexed competitive binding assay that uses a cocktail solution of labeled ligands with a GPCR microarray.
  • three kinds of receptors one in each column of microspots, are immobilized on a support surface in an array.
  • a "cocktail" solution containing three labeled-ligands with a test compound.
  • the ligands A and B corresponding to receptors A and B, are labeled using fluorophores that are either identical or similar in terms of their spectral fluorescence signature.
  • the ligand corresponding to receptor C is labeled with a different kind of fluorophore that exhibits an excitation and emission spectrum distinguishable from the fluorophore(s) labeling ligands A and B.
  • the cocktail solution is applied, two possible situations are depicted. In the first situation, no specific inhibition is observed since the test compound does not bind to any of the receptor proteins. In the second situation, we observe a reduction in signal intensity due to specific inhibition from the test compound to the binding of the paired labeled-ligand to its cognate receptor C. Thus, the test compound specifically inhibits binding to receptor C.
  • the benefits of the present invention encourage multiplexing and miniaturization, which are particularly pertinent for today, due to the increased pace and need for rapid target identification.
  • Binding assays generally are directed to protein/peptide profiling uses. Current examples of such assays involve immobilized protein molecules of interest on a surface at defined locations. (See, Wilson, D.S. and Nock, S., "Functional Protein Microarrays," Curr. Opinion in Chemical Biology 2001, 6, 81-85; Zhu, H., et ah,
  • Pirrung et al. The methods developed by Pirrung et al. have not taken account the complexity associated with multiplexed applications as outlined above, and likely are not feasible for such purposes. Further, their platform is mainly constructed for a direct binding profile assay of a peptide/protein in a sample.
  • the present invention involves a indirect binding assay, which takes advantage of the competitive ability of small biological, biochemical or chemical molecules or compounds (e.g., molecular weight ⁇ -10,000 daltons, preferably between about 100-5000 daltons) to bind with receptors on the array against the predetermined labeled-ligands's ability to bind with the same receptors.
  • Pirrung et ⁇ /.'s platform relies on a design in which the number of the probe polypeptide(s) in the microarray overwhelms the number of available target polypeptide molecules to be detected in a sample.
  • the present invention takes advantage of a reversible binding system in which the labeled ligands binds to their paired probe receptors in the microarray.
  • the probe receptors can be, and are preferably (profiling or screening tests), saturated by the binding of labeled ligands. From a combinatorial "numbers" perspective, attempting to map out biological target space for a group of proteins (e.g.
  • Thematic GPCR Microarrays Unlike tens of thousands of genes one has to consider for array design in DNA microarrays, there is much smaller number of GPCRs - the human genome contains about 400-700 GPCRs. Scientitist have discovered ligands associated with about 200 GPCRs. Receptors whose natural agonist remains unidentified are referred to as "orphan" GPCRs. hi one embodiment, all of the known and/or orphan GPCRs could be arrayed on a surface to form a full index array; such arrays can be used for target identification, natural agonist identification and compound screening.
  • one subfamily member of each subfamily of all GPCRs can be arrayed together on a surface to form a representative index array; such arrays can be used for compound screening, and more suitable for classifying compounds against family GPCRs.
  • GPCRs in the same organ, tissue or even single cell is an extremely important factor to be considered and monitored during drag development.
  • HTS techniques are related to single target screening at one time.
  • GPCR arrays can be used to evaluate the selectivity of multiple compounds of interest to a variety of receptors.
  • members of a single or several related subfamilies of GPCRs can be arrayed on a surface; such arrays are preferably for compound pharmacological profiling and selectivity screening.
  • Figures 2A-C show an enlarged, exploded view of the bottom of microtiter well plate (a.k.a., microplate).
  • Figure 2A is the overview of the microplate.
  • Figure 2B depicts a small set of pre-selected GPCRs associated, preferably fluidly, or embedded in lipid membranes.
  • the membranes are deposited and immobilized at defined locations on the bottom surface of a well in the microplate forming an array of microspots.
  • Figure 2C illustrates, as an idea orientation, the lipid-membrane containing the GPCR is immobilized and arranged with the N-terminal-side of the receptor protein molecule directed away from the solid support of the well-bottom.
  • the membranes are deposited and immobilized at defined locations on the bottom surface of a well in the microplate forming an array of microspots.
  • Figure 2C illustrates, as an idea orientation, the lipid-membrane containing the GPCR is immobilized and arranged with the N-terminal-side of the receptor protein molecule directed away from the solid support of the well-bottom.
  • the lipid-membrane containing the GPCR is immobilized and arranged with the N-terminal-side
  • GPCR molecules in the membranes are coupled with certain trimeric G-protein.
  • GPCRs that share similar specific tissue distribution, or specific roles in physiology and pharmacology are arrayed on a surface; such arrays are preferred for compound selectivity screening.
  • Some GPCRs are preferably distributed in certain types of tissues.
  • some receptors including the muscarinic acetylcholine receptor, dopamine 2 receptor, histamine 2 receptor, serotonin 4 receptor and prostaglandin receptor prominently distribute in the gastrointestinal system, while some receptors including serotonin 1A/1D and 2A/2C receptor, neurotensin 1 and 2 receptors, opioid receptors (mu, delta, kappa, ORL-1), and dopamine 2/3 receptors prominently distribute in the central nervous system
  • a GPCR and its physiologically important or random mutants are arrayed on a surface; such arrays are preferably suitable for studying structure-activity relationships, as well as screening highly specific drag compounds for a GPCR mutant that plays a key role in a given human disease or cancer. Some GPCRs and their mutants are related to the development of certain tumors.
  • rhodopsin are related to retinitis pigmentosa
  • vasopressin V2 are related to X-linked nephrogenic diabetes (Stadel, J. M., et al., Trends in Pharamaco. Sciences 1997, v. 18, 430-437).
  • the GPCRs of personal interest can be arrayed on a surface; such custom arrays can be used to serve their own selectivity screening of compounds.
  • the ligand can be selected from a group including: antagonists, agonists, partial-agonists, or inverse- agonists.
  • the ligand also may be a naturally occurring agonist or a synthetic chemical, biochemical, or biological compound or molecule.
  • the ligand can be a nucleotide, a nucleoside, a modified nucleotide or nucleoside, an organic or inorganic compound, a peptide, a polypeptide or protein, a lipid, or a modified lipid.
  • the ligand should bind readily to a corresponding receptor protein and may be labeled.
  • the ligand when it is labeled, should be detectable by means of a variety of state-of-the-art techniques or methodologies.
  • the label is selected based on the desired detection technology employed, including fluorescence, radioactive detection, chemical or bioluminescence, or phosphor up-conversion, or other technologies.
  • the label is a fluorescent dye (e.g., Bodipy-fluorescein, Bodipy-TMR., Rhodamine, Texas- Red, Cy-3, Cy-5, or fluorescein).
  • the label is a radio-isotope, such as tritium or P 32 , S 35 , or I 125 .
  • the detection technique can be a labeled streptavidin, anti-biotin antibody, or a streptavidin or anti- biotin antibody-coated particle (e.g., gold nanoparticle, wherein the resonant light scattering may be employed for detection).
  • a direct-binding assay based on an evanescent-field detector employing a grating-coupled waveguide, surface-plasmon resonance, or other mass-based biosensor system, can be used, hi this situation, the ligand can be either unlabeled or labeled.
  • a label can be a species that may have a sufficient mass or an optical charactertistic useful for enhanced detection sensitivity.
  • a gold particle with a diameter of about 1 nanometer to about 45 nanometers, or up to about 100 nanometers can be attached to a ligand.
  • a labeled ligand should satisfy several critical properties, as mentioned above.
  • An ideal fluorescently labeled ligand for GPCR microarray applications should be relatively hydrophihc, have low net charge, have good photostability, and have a binding affinity (K d ) of several nanomolar with a specificity of greater than or equal to about 55% or 60% for its paired receptor in the array.
  • the ligand should also exhibit minimal cross-activity to other kinds of receptors.
  • Labeled-Ligand Cocktail Solution Generally, according to the invention, a cocktail solution containing more than one labeled ligand is used in multiplexed binding assays. In the cocktail solution, each labeled ligand should bind only to its paired GPCR(s) in an array.
  • Cy5- naltrexone works as a labeled ligand for delta2 opioid receptor, because Cy 5 -naltrexone is more hydrophihc than fluorescein-naltrexone (e.g., commercially available from the Molecular Probes, Inc.), can bind to delta2 opioid receptor with a K d of 2.5 nM with a specificity of -90% at the concentration of one to four fold (l-4x) of K , and has minimal cross activity to a number of receptors, such as neurotensin receptor (NTRl), neurokinin receptor subtype 2 (NK2), motilin receptor (MOTR), and betal, beta2, and alpha2A adrenergic receptors.
  • NTRl neurotensin receptor
  • NK2 neurokinin receptor subtype 2
  • MOTR motilin receptor
  • Bodipy-TMR-motilin 1-16 (BT-MOT 16) is chosen as a labeled ligand for motilin receptor, because BT-MOT16 is relatively hydrophilic and has neutral charge at physiological pH, can bind to the MOTR with a K of -3 nM with a specificity of -75% at the concentration of l-4x K d , and has minimal cross activity to a number of receptor, such as neurotensin receptor (NTRl), delta2 opioid reeceptor, neurokinin receptor subtype 2 (NK2), and betal, beta2, and alpha2A adrenergic receptors.
  • NTRl neurotensin receptor
  • NK2 neurokinin receptor subtype 2
  • beta2A adrenergic receptors such as beta2, and alpha2A adrenergic receptors.
  • Bodipy-TMR-CGP 12177 has been reported to bind specifically to the ⁇ l and ⁇ 2 adrenergic receptors with similar affinity (K d of -1-2 nM).
  • K d affinity
  • et al J. Am. Chem. Soc. 2002, 124, 2394-2395
  • Baker, J.G., et al Brit. J. Pharmacol. 2002, 139, 232-242.
  • Labeled ligands in the cocktail could be agonists and/or antagonists; the concentration of each ligand in the cocktail should be relatively close to the value of the K d of the ligand to the paired receptor(s) in order to maximize total binding signal(s) as well as specificity.
  • concentration of labeled ligand can be about - 0.7 or 1 to 4 fold of the K d value.
  • Labeled ligands in the cocktail can be labeled with different fluorescent dye moieties (e.g., rhodamine-, Bodipy-TMR-, Cy3-, Cy5-, etc); the detection can be multi-channel fluorescence scanner (e.g, FITC, Cy3,
  • Multiplexed Saturation Assay for Parallel K Determination Multiplexed saturation assay can be used to determine binding constants (i.e.,
  • K d K d ) of labeled ligands to their paired receptors in parallel.
  • a cocktail of labeled ligands are used; each labeled ligand binds specifically to its own paired GPCR in the arrays with no or minimal cross activity to other receptors in the same arrays.
  • Two subsets of microarrays were incubated individually with a buffered solution containing labeled ligand cocktail, each at different concentrations in the absence and presence of their unlabeled counteipart ligand in excess.
  • Compound Multiplexed competitive binding assays can be used to determine "selective potency" of a single compound against each labeled ligand in the cocktail to its own paired GPCR in the microarrays.
  • a cocktail of labeled ligands is used; each labeled ligand binds specifically to its own paired GPCR in the arrays with no or minimal cross activity to other receptors in the same arrays.
  • multiple sub- arrays are treated individually with a given compound at different concentrations in the presence of the labeled ligand cocktail, each at a predetermined, constant concentration. The fluorescence intensity of each receptor microspot was examined as a function of the concentration of the compound, and an IC 5 o value was later extracted.
  • FIGS. 4A-C represent the results of an experiment conducted according to an embodiment of present method.
  • Figure 4A shows concentration dependent inhibition profile of the binding reaction between Cy5 -naltrexone and OPl -receptor by naltrexone.
  • Figures 4B and 4C respectively, show the concentration dependent inhibition profiles of the binding reaction of BT-MOT16 with MOTR, and Cy5-NT with NTRl.
  • concentration of each of the three labeled ligands in the cocktail solution is: BT-MOT 16, 5 nM; Cy5-NT, 5 nM; and Cy5- naltrexone, 2.5 nM.
  • Multiplexed competitive binding assays can be used to determine relative potency of multiple compounds against the binding of each labeled ligand in the cocktail solution to its paired corresponding GPCR in a microarray.
  • a concentration range employed e.g., up to micromolar concentrations, using alternative methods or by using an initial screen using GPCR microarrays, as illustrated in Figure 6) with minimal cross-binding to other receptors in the same array.
  • a cocktail solution of labeled ligands is used.
  • Each labeled ligand binds specifically to its own paired GPCR in the array with little or no cross activity to other receptors in the same arrays.
  • the sub-arrays are treated separately and individually with a solution of unlabeled compounds at different concentrations in the presence of the cocktail solution of labeled ligands, each of which are at a fixed concentration.
  • the fluorescence intensity of each receptor microspot is examined as a function of the concentration of the cocktail of compounds, and IC50 values for the compounds are estimated.
  • Two sets of experiments are carried out. The first set involves using three compounds (neurotensin for NTRl, motilin for MOTR, and naltrexone for delta2).
  • the second set involves using three other compounds (neuromedin N to NTRl, endomorphin II to delta2, motilin for MOTR) .
  • three other compounds neuropeptides, endomorphin II to delta2, motilin for MOTR.
  • the relative potencies of these compounds against the binding affinity of Cy5 -naltrexone to delta2, Cy5-NT to NTRl, and BT-MOT 16 to MOTR are examined. The results are summarized graphically in accompanying Figures 5A-C.
  • Multiplexed competitive binding assays for Compound Screening using Cocktail Solution of Labeled-Ligands.
  • Multiplexed competitive binding assays can be used for screening compounds which potentially can inhibit the binding of at least one labeled ligand in the cocktail solution with their paired GPCRs.
  • a cocktail solution of labeled ligands is provided.
  • Each labeled ligand binds specifically to its own corresponding GPCR in the arrays with minimal or no cross activity with other receptors in the same array.
  • each sub-array is treated individually with at lease one compound at a fixed concentration (generally - 1 ⁇ M) in the presence of a fixed concentration of the cocktail solution of labeled ligands.
  • multiplexed Competitive Binding Assays for Compound Screening Using a Single Labeled-Ligand one may employ a single labeled ligand that can specifically bind with at lease two receptors in the same sub-arrays with desired affinities.
  • a single labeled ligand that can specifically bind with at lease two receptors in the same sub-arrays with desired affinities.
  • An alternative multiplexed binding assay for compound screening involves using fluorescently labeled ligands that are pre-bound to GPCR molecules.
  • An assay of this format involves incubating the GPCR microarray with the labeled ligands prior to treatment with solutions containing putative ligands. This kind of assay is feasible because of the high affinity of labeled ligands to their paired GPCRs.
  • the binding event is essentially irreversible over the duration of the assay, since mass transport limited rebinding greatly reduces the rate of dissociation of labeled ligands.
  • Treatment of the array with solutions containing a competitive ligand can effect the displacement of a bound ligand. Data for an example is summarized in the images of Figures 8A-D.
  • the present invention also describes a method for increasing the overall multiplexing capability of a microarray. Such a method can be particularly useful for microarrays located in a microtiter plate, which have multiple biological probes arrayed onto the bottom of either 96-well or 384- well microtiter plates. Due to the spatial constraints on the bottom surface of a microplate well, only a limited number of biological probes may be immobilized and analyzed at one time. For example, in a
  • 384- well plate only - 0.136 cm 2 is available for depositing an array.
  • each well in 384- well microtiter plate can only be used to array 3-4 receptors, each in triplicate; or for a maximum of about 20 elements per well.
  • This limited number of elements is typically insufficient for high tliroughput screening uses, which have a selectivity panel of probe receptors, ideally numbering in the range of about 5-10, with each kind of probe in the array in triplicate or quadruplicate, for statistical reliability.
  • the method involves the use of at least two receptors co-immobilized within a single microspot, in combination with at least a two-color detection approach enabling at least a doubling of the capacity of probe elements for biological microarrays.
  • the new approach is described in terms of GPCR microarrays, the general concept is not necessarily limited to just one species or kind of microarray application. Any type of biological microarray which uses probe elements can benefit from the present method.
  • at least two biological targets are mixed together and the combined mixture is used to fabricate microarrays.
  • the method exploits a system with at least two-color detection.
  • a two-color detection system employs a cocktail of two mixed labeled-ligands with different label tags, and each labeled ligand specifically binds to only one probe in each microspot.
  • the tags can be different in terms of physical and chemical properties.
  • these tags are fluorescence dyes.
  • these tags are nano-particles that give rise to different mass or light emitting (scattering) properties. Since most current fluorescence imaging technologies (e.g., FITC, Cy3, Texas Red, and Cy5) are limited to four channels, four probes can be set in each microspot.
  • the present invention relates to a thematic GPCR microarray and methods for profiling or screening ligands or compounds.
  • the thematic multiplexed GPCR microarray comprises: a plurality of GPCRs arranged on a substrate at positionally defined locations; said GPCRs are either a) a member from each subfamily of GPCRs, or b) at least a single member selected from several related subfamilies of GPCRs, or c) a GPCR and its mutants or its corresponding GPCRs originated from different species.
  • the GPCRs in said microarray are related to each other by either: functionality, physiology, family, pathology, tissue-distribution, or mutagenesis, or evolutionary history.
  • the method comprises: a) providing an plurality of receptor microspots on a substrate to form an array; b) preparing a cocktail solution of labeled ligands, each labeled ligand having an affinity to bind with at least one corresponding paired receptor in said array; wherein said cocktail solution contains either 1) only labeled ligands, each at a different concentration, or 2) labeled ligands, each at a different concentration, in the presence of counterpart ligands in excess; c) contacting said cocktail solution with said array; and d) determining the binding affinity of each labeled ligand to its said paired receptor.
  • the receptor is a guanine nucleotide- binding protein-coupled receptor (GPCR), a ligand-gated ion channel receptor, a tyrosine kinase receptor, serine/threonine kinase receptor, or guanylate cyclase receptor.
  • GPCR guanine nucleotide- binding protein-coupled receptor
  • the GPCR is associated with a biological membrane, which may be either i) a cell membrane fragment containing a GPCR, or ii) a liposome or micell containing a reconstituted GPCR.
  • the labeled-ligand in said cocktail solution is a chemical molecule or biological molecule that can bind readily to a receptor with a specific binding affinity constant.
  • a method profiling or screening a compound or target comprises: a) providing an plurality of receptor microspots on a solid surface to form an array; b) preparing a cocktail solution of labeled ligands, each labeled ligand having an affinity to bind with at least one corresponding paired receptor in said array; wherein a compound or multiple compounds are either present or absent from said cocktail solution; c) contacting said cocktail solution with said array; and d) determining the potency of said compound against the binding of each labeled ligand to its said paired receptor.
  • the method comprises: a) providing an plurality of receptor microspots on a solid surface to form an array; b) preparing a cocktail solution of labeled ligands, each labeled ligand having an affinity to bind with at least one corresponding paired receptor in said array; c) contacting said cocktail solution with said array; d) determining the total binding signal of each receptor; e) sequentially contacting said array with a second solution containing a compound; and f) determining the amount of pre-bound labeled ligands to each receptor displaced by said compound.
  • the method comprises: a) providing an plurality of GPCR microspots on a solid surface to form an array; b) preparing a cocktail solution of labeled ligands, each labeled ligand having an affinity to bind with at least one corresponding paired GPCR in said array, in either the absence or presence of a compound; c) contacting said cocktail solution with said array; and d) determining the binding profile of the compound against the labeled ligand to its said paired receptor.
  • the method to screen or profile a compound comprises: a) providing an plurality of receptor microspots on a solid surface to form an array; b) preparing a solution containing at least one compound; c) contacting the array with the solution; d) detecting the binding of the compound to said receptors; and c) determining the binding profile of said compound to its said paired receptor.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Endocrinology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un microdosage thématique et des procédés destinés aux dosages de liaison multiplexés utilisant une solution cocktail de ligands étiquetés en présence ou en absence d'un composé cible. Les procédés permettent aux chercheurs de cribler les composants à la recherche de cibles multiples en utilisant un format de microdosage.
PCT/US2004/019750 2003-07-11 2004-06-21 Dosages de liaison multiplexes destines a des reseaux de recepteurs WO2005010532A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2006520183A JP2007530919A (ja) 2003-07-11 2004-06-21 受容体アレイ用の多重化結合アッセイ
EP04776836A EP1646871A1 (fr) 2003-07-11 2004-06-21 Dosages de liaison multiplexes destines a des reseaux de recepteurs

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US48659203P 2003-07-11 2003-07-11
US60/486,592 2003-07-11
US10/639,718 US20050009204A1 (en) 2003-07-11 2003-08-12 Multiplexed binding assays for receptor arrays
US10/639,718 2003-08-12

Publications (1)

Publication Number Publication Date
WO2005010532A1 true WO2005010532A1 (fr) 2005-02-03

Family

ID=33567930

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/019750 WO2005010532A1 (fr) 2003-07-11 2004-06-21 Dosages de liaison multiplexes destines a des reseaux de recepteurs

Country Status (4)

Country Link
US (1) US20050009204A1 (fr)
EP (1) EP1646871A1 (fr)
JP (1) JP2007530919A (fr)
WO (1) WO2005010532A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005066633A3 (fr) * 2003-12-19 2006-02-16 Corning Inc Ligands susceptibles de fluorescence pour matrices de recepteurs couples a la proteine g
WO2006066368A1 (fr) * 2004-12-23 2006-06-29 Vrije Universiteit Brussel Procede de criblage et de selection de ligands
JP2008224235A (ja) * 2007-03-08 2008-09-25 Nec Corp 創薬のマルチターゲットスクリーニング装置
JP2010504098A (ja) * 2006-09-21 2010-02-12 ユニバーシティー オブ ロチェスター 筋緊張性ジストロフィーのためのタンパク質置換治療に関する組成物および方法
US8080408B2 (en) 2006-07-14 2011-12-20 Sony Corporation Bioassay substrate and bioassay method
US10458993B2 (en) 2012-06-01 2019-10-29 Heptares Therapeutics Limited Assay for assessing conformational stability of membrane protein
US10766945B2 (en) 2011-08-10 2020-09-08 Heptares Therapeutics Limited Stable proteins
US11673938B2 (en) 2007-03-22 2023-06-13 Heptares Therapeutics Limited Mutant G-protein coupled receptors and methods for selecting them

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006078814A2 (fr) * 2005-01-20 2006-07-27 The Regents Of The University Of California Microreseaux cellulaires pour le criblage de facteurs de differenciation
US20060183166A1 (en) * 2005-02-11 2006-08-17 Michael Mayer Arrays of supported biomembranes and uses thereof
US20090131263A1 (en) * 2007-11-19 2009-05-21 Longying Dong Normalization methods for G-protein coupled receptor membrane array
GB0724860D0 (en) * 2007-12-20 2008-01-30 Heptares Therapeutics Ltd Screening
US20100130829A1 (en) * 2008-11-25 2010-05-27 Ye Fang Materials and methods for detecting early cell migration events
JP6059014B2 (ja) 2009-06-19 2017-01-11 アリゾナ ボード オブ リージェンツ ア ボディー コーポレート アクティング オン ビハーフ オブ アリゾナ ステイト ユニバーシティＡＲＩＺＯＮＡ ＢＯＡＲＤ ＯＦ ＲＥＧＥＮＴＳ，ａ ｂｏｄｙ ｃｏｒｐｏｒａｔｅ ａｃｔｉｎｇ ｏｎ ｂｅｈａｌｆ ｏｆ ＡＲＩＺＯＮＡ ＳＴＡＴＥ ＵＮＩＶＥＲＳＩＴＹ 試料の構成成分の検出において使用されるデバイスを製造する方法およびペプチドアレイ
EP3008083B1 (fr) 2013-06-11 2021-06-30 Paul Scherrer Institut Procédé de détermination de liaison ligand mutable-gpcr à une résolution d'un seul acide aminé et paires de ligand muté et de gpcr
DE102015011134A1 (de) * 2015-08-31 2017-03-02 Thyssenkrupp Ag Raststern für eine Kraftfahrzeuglenksäule und Verfahren zur Herstellung dieses
US10758886B2 (en) 2015-09-14 2020-09-01 Arizona Board Of Regents On Behalf Of Arizona State University Conditioned surfaces for in situ molecular array synthesis
WO2017223116A2 (fr) 2016-06-20 2017-12-28 Healthtell Inc. Procédés pour le diagnostic différentiel de maladies auto-immunes
CA3028975A1 (fr) 2016-06-20 2017-12-28 Healthtell Inc. Methodes pour le diagnostic et le traitement de maladies auto-immunes
WO2018089858A1 (fr) 2016-11-11 2018-05-17 Healthtell Inc. Procédés pour l'identification de biomarqueurs candidats
WO2019055618A1 (fr) 2017-09-15 2019-03-21 Arizona Board Of Regents On Behalf Of Arizona State University Procédés de classification des réponses à une immunothérapie anticancéreuse
CN114467031A (zh) * 2019-10-01 2022-05-10 Ac免疫有限公司 用于配体筛选的微放射性结合测定
EP4038222A4 (fr) 2019-10-02 2023-10-18 Arizona Board of Regents on behalf of Arizona State University Procédés et compositions pour identifier des néo-antigènes destinés à être utilisés dans le traitement et la prévention du cancer

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999064436A1 (fr) * 1998-06-12 1999-12-16 Merck & Co., Inc. Clonage et identification du recepteur de la motiline
WO2000063701A2 (fr) * 1999-04-15 2000-10-26 The Board Of Trustees Of The Leland Stanford Junior University Jeux ordonnes de microechantillons de polypeptides
US20020094544A1 (en) * 2000-08-10 2002-07-18 Ye Fang Arrays of biological membranes and methods and use thereof

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1339723C (fr) * 1988-01-19 1998-03-17 Philip Mcmahon Immunoesssai a point d'essai multiples
US5225326A (en) * 1988-08-31 1993-07-06 Research Development Foundation One step in situ hybridization assay
US6699719B2 (en) * 1996-11-29 2004-03-02 Proteomic Systems, Inc. Biosensor arrays and methods
EP2295988A2 (fr) * 1996-12-31 2011-03-16 High Throughput Genomics, Inc. Dispositif d'analyse moléculaire multiplexé et procédé de sa fabrication
US6087102A (en) * 1998-01-07 2000-07-11 Clontech Laboratories, Inc. Polymeric arrays and methods for their use in binding assays
US6569383B1 (en) * 2000-03-11 2003-05-27 Intrinsic Bioprobes, Inc. Bioactive chip mass spectrometry
US6977155B2 (en) * 2000-08-10 2005-12-20 Corning Incorporated Arrays of biological membranes and methods and use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999064436A1 (fr) * 1998-06-12 1999-12-16 Merck & Co., Inc. Clonage et identification du recepteur de la motiline
WO2000063701A2 (fr) * 1999-04-15 2000-10-26 The Board Of Trustees Of The Leland Stanford Junior University Jeux ordonnes de microechantillons de polypeptides
US20020094544A1 (en) * 2000-08-10 2002-07-18 Ye Fang Arrays of biological membranes and methods and use thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FANG Y ET AL: "G-PROTEIN-COUPLED RECEPTOR MICROARRAYS", CHEMBIOCHEM - A EUROPEAN JOURNAL OF CHEMICAL BIOLOGY, WILEY VCH, WEINHEIM, DE, vol. 3, no. 10, 4 October 2002 (2002-10-04), pages 987 - 991, XP001155243, ISSN: 1439-4227 *
FANG Y ET AL: "MEMBRANE PROTEIN IN MICROARRAYS", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, AMERICAN CHEMICAL SOCIETY, WASHINGTON, DC, US, vol. 124, no. 11, 20 March 2002 (2002-03-20), pages 2394 - 2395, XP001156134, ISSN: 0002-7863 *
FANG YE ET AL: "G protein-coupled receptor microarrays for drug discovery.", DRUG DISCOVERY TODAY, vol. 8, no. 16, 15 August 2003 (2003-08-15), pages 755 - 761, XP002305895, ISSN: 1359-6446 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005066633A3 (fr) * 2003-12-19 2006-02-16 Corning Inc Ligands susceptibles de fluorescence pour matrices de recepteurs couples a la proteine g
WO2006066368A1 (fr) * 2004-12-23 2006-06-29 Vrije Universiteit Brussel Procede de criblage et de selection de ligands
EP2327780A1 (fr) * 2004-12-23 2011-06-01 Vrije Universiteit Brussel Procédé pour le tri et la séléction de ligands
US8080408B2 (en) 2006-07-14 2011-12-20 Sony Corporation Bioassay substrate and bioassay method
JP2010504098A (ja) * 2006-09-21 2010-02-12 ユニバーシティー オブ ロチェスター 筋緊張性ジストロフィーのためのタンパク質置換治療に関する組成物および方法
JP2014055983A (ja) * 2006-09-21 2014-03-27 Univ Of Rochester 筋緊張性ジストロフィーのためのタンパク質置換治療に関する組成物および方法
US9371527B2 (en) 2006-09-21 2016-06-21 University Of Rochester Compositions and methods related to protein displacement therapy for myotonic distrophy
JP2008224235A (ja) * 2007-03-08 2008-09-25 Nec Corp 創薬のマルチターゲットスクリーニング装置
US11673938B2 (en) 2007-03-22 2023-06-13 Heptares Therapeutics Limited Mutant G-protein coupled receptors and methods for selecting them
US10766945B2 (en) 2011-08-10 2020-09-08 Heptares Therapeutics Limited Stable proteins
US10458993B2 (en) 2012-06-01 2019-10-29 Heptares Therapeutics Limited Assay for assessing conformational stability of membrane protein

Also Published As

Publication number Publication date
JP2007530919A (ja) 2007-11-01
US20050009204A1 (en) 2005-01-13
EP1646871A1 (fr) 2006-04-19

Similar Documents

Publication Publication Date Title
US20050009204A1 (en) Multiplexed binding assays for receptor arrays
EP1388010B1 (fr) Reseaux de microdepots en membranes biologiques
Fang et al. G protein-coupled receptor microarrays for drug discovery
US6977155B2 (en) Arrays of biological membranes and methods and use thereof
Heilker et al. G-protein-coupled receptor-focused drug discovery using a target class platform approach
Gupta et al. Targeting opioid receptor heterodimers: strategies for screening and drug development
US7816096B2 (en) Assay solution compositions and methods for GPCR arrays
Drakopoulos et al. Development and biological applications of fluorescent opioid ligands
Waller et al. Techniques: GPCR assembly, pharmacology and screening by flow cytometry
WO2005121755A1 (fr) Analyse acellulaire permettant d'identifier le recepteur couple a la proteine g et son ligand
Waller et al. High throughput screening of G-protein coupled receptors via flow cytometry
Venton et al. Screening combinatorial libraries
WO2001050127A2 (fr) Methode permettant d'identifier des ligands de molecules biologiques cibles
Fang et al. Membrane biochips
Fu et al. The identification of high-affinity G protein-coupled receptor ligands from large combinatorial libraries using multicolor quantum dot-labeled cell-based screening
Sklar et al. High-Throughput Flow Cytometry
Greasley et al. G-protein-coupled receptor screening technologies
Saliba Drug Improvement and Design Involving G-Protein Coupled Receptors
WO2005066633A2 (fr) Ligands susceptibles de fluorescence pour matrices de recepteurs couples a la proteine g
Karimian Doktorin der technischen Wissenschaften
Karimian Subcellular micropatterning for spatio-temporal characterization of protein-interactions in the live cell membrane/Author Tina Karimian
Jackson et al. Microarrays meet the Voltaire challenge: Drug discovery on a chip?
US20150204890A1 (en) Microarrays of g protein coupled receptors
Fang et al. G-Protein Coupled Receptor Microarrays: A Versatile Tool for Drug
Salon et al. 18 G Protein–Coupled Receptor Microarrays for Drug Discovery

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2006520183

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2004776836

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2004776836

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载